Organization and structure of plastome psbF,psbL, petG and ORF712 genes in Chlamydomonas reinhardtii

Summary We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement. psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b₅₅₉ locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5-and 3-ends to the ribosomal protein rps3 gene, but contains, a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.     

rps10, unreported for plastid DNAs,is located on the cyanelle genome of Cyanophora paradoxa and is contranscribed with the str operon genes

Summary rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the S10 operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.     

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

Summary The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5 and 3 ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3. Abreviations: Gene names follow the convention of Hallick and Bottomley (1983): rp123, rpl2, rpl22 are, respectively, genes for the L23, L2, L22 polypeptides of the 50S ribosomal subunit; rps19 and rps3 are genes for the S19 and S3 polypeptides of the 30S ribosomal subunit     

The chloroplast gene cluster containing psbF,psbL, petG and rps3 is conserved in Chlamydomonas

We have sequenced a 6.8-kb segment of the Chlamydomonas eugametos chloroplast DNA which contains the psbF, psbL, petG and rps3 genes. As in the distantly related green alga Chlamydomonas reinhardtii, these genes reside in this order (53) on the same DNA strand, suggesting that such a chloroplast gene cluster was present in the most recent common ancestor of all Chlamydomonas species. For each of the four genes, with the exception of rps3, the C. eugametos and C. reinhardtii coding regions were found to be identical, or very similar, in length, whereas each of the intergenic spacers is substantially longer in C. eugametos than in C. reinhardtii. The central portion of both Chlamydomonas rps3 genes features a long extra coding region relative to other rps3 sequences. We have shown that the insertion sequence in the C. eugametos rps3 is not excised at the RNA level.     

The gene structure of B-type nuclear lamins ofXenopus laevis: Implications for the evolution of the vertebrate lamin family

The gene structure of theXenopus laevis lamin B1 gene is presented, together with a partial analysis of the lamin B2 gene of the same species. The intron/exon pattern of lamin B1 is strikingly similar to other vertebrate lamin genes and is completely identical to that ofXenopus lamin B3. An additional intron present in the murine lamin B2 gene has a counterpart in the orthologue gene ofXenopus. These data complete a comparison of the genomic organization of all vertebrate lamin types known so far. They allow the conclusion that the genes encoding B1 and B3 probably reflect the ancestral lamin gene organization and that the B2-type lamins are derived from them. A non-conforming splice junction sequence is found in theXenopus lamin B1 gene. A GC is found instead of the canonical GT dinucleotide at the 5 end of intron IX. Comparison with other unusual splice sites indicates that the lamin B1 gene represents a functional gene.     

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

Summary Nucleotide sequence analysis of a 17043 basepair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5-and 3-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5 half of rpoC (termed rpoC1 in other species) is not present at the 5 end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.     

Splicing and editing of rps10 transcripts in potato mitochondria

The structure and expression of the potato mitochondrial gene rps10, encoding ribosomal protein S10, has been characterized. The RPS10 polypeptide of 129 amino acids is encoded by two exons of 307 bp and 80 bp respectively, which are separated by a 774-bp class-II intron. Editing of the complete rps10 coding region was studied by sequence analysis of spliced cDNAs. Four C residues are edited into U, resulting in the creation of a putative translational initiation codon, a new stop codon which eliminated ten carboxy-terminal residues, and two additional amino-acid alterations. All these changes increase the similarity between the potato and liverwort polypeptides. One additional C-to-U RNA editing event, observed in the intron sequence of unspliced cDNAs, improves the stability of the secondary structure in stem I (i) of domain I and may thus be required for the splicing reaction. All spliced cDNAs, and most unspliced cDNAs, were completely edited, suggesting that editing is an early step of rps10 mRNA processing and precedes splicing. Earlier work on potato rps10 (Zanlungo et al. 1994) is now known to comprise only a partial analysis of the gene, since the short downstream exon was not identified.     

A chloroplast DNA mutational hotspot and gene conversion in a noncoding region near rbcL in the grass family (Poaceae)

The noncoding DNA region of the chloroplast genome, flanked by the genes rbcL and psaI (ORF36), has been sequenced for seven species of the grass family (Poaceae). This region had previously been observed as a hotspot area for length mutations. Sequence comparison reveals that short duplications, probably resulting from slipped-strand mispairing, account for many small length differences between sequences but that major mutational hotspots are localized in three small areas, two of which show potential secondary structure. Mutation in one of these hotspots appears to be a result of more complex recombination events. All seven species contain a pseudogene for rpl23 and evidence is presented that this pseudogene is being maintained by gene conversion with the functional gene. Different transition/transversion biases and AT contents between the pseudogene and the surrounding noncoding sequences are noted. In the subfamily Panicoideae there is a deletion in which almost 1 kb of ancestral sequence, including the 3 end of the rpl23 pseudogene, has been replaced by a non-homologous 60-base sequence of unknown origin. Two other deletions of almost the same region have occurred in the grass family. The deleted noncoding region has mutational and compositional properties similar to the rbcL coding sequence and the rpl23 pseudogene. The three independent deletions, as well as the pattern of mutation in the localized hotspots, indicate that such noncoding DNA may be misleading for studies of phylogenetic inference.     

Loss of all plastid ndh genes in Gnetales and conifers: extent and evolutionary significance for the seed plant phylogeny

The exact phylogenetic position of Gnetales, a small, highly modified group of gymnosperms with an accelerated rate of molecular evolution, is one of the most challenging issues for seed plant systematics. Recent results from entire plastid genome (ptDNA) sequencing revealed the absence of the entire suite of plastid ndh genes in several species of Gnetales and the pine family (Pinaceae) potentially highlighting a major structural feature linking these two groups—concerted loss of all plastid genes for the NADH dehydrogenase complex. However, the precise extent of ndh gene loss in gymnosperms has not been surveyed. Using a slot-blot hybridization method, we probed all 11 ndh genes in 162 species from 70 of 85 gymnosperm genera. We find that all ndh genes are absent across Gnetales and Pinaceae, but not in any other group of gymnosperms. This feature represents either a major synapomorphy for a clade consisting of these two lineages or, less likely, a convergent loss. Our survey substantially extends previous inferences based on more limited sampling and, if the former evolutionary interpretation is correct, it provides additional support for the contentious “gnepine” hypothesis, which places Gnetales as sister to Pinaceae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Localization of seed protein genes on flow-sorted field bean chromosomes

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using nested primers. The genes are located within defined regions of chromosome I (USP=unknown seedprotein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes 1) and V (legumin A genes and pseudogenes 1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.     

Restriction and gene maps of plastid DNA from Capsicum annuum

Summary Chloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to chromoplasts in Capsicum annuum does not involve any large rearrangements of the plastid chromosome. A restriction map of Capsicum annuum plastid DNA was constructed using the same restriction enzymes in single digests and in all possible pair combinations. Overlapping restriction fragments were identified by digesting each product of a single digest with each of the other three enzymes. The resulting restriction map is similar to that of chloroplast DNA from other members of the Solanaceae with respect to most restriction sites. The genome size corresponds to 143 kbp. The locations of 24 genes, coding for ribosomal RNAs and for proteins of Photosystem I (PSI), Photosystem II (PSII), ATP synthase, cytochromes, the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) (RuBPC), and ribosomal proteins were determined by probing Southern blots of Capsicum chloroplast DNA with probes of genes from spinach and tobacco. The gene locations are completely conserved with respect to those of other members of the Solanaceae and the majority of higher land plants.Abbreviations and notations atpA, atpB, atpE, and atpF genes for the , , , and I subunit, respectively, of ATP synthase - cpDNA chloroplast DNA - petA, petB, petD genes for cytochrome f, cytochrome b ₆, and subunit IV of cytochrome b ₆/f complex, respectively - psaA, psaB, psaC genes for the P₇₀₀ apoproteins - psbA gene for Q_B - psbB and psbC genes for the 51-kDa and 44-kDa proteins, respectively, of PSII - psbD gene for the Q_B-like polypeptide of PSII - psbE gene for cytochrome b ₅₅₉ - rbcL gene for the large subunit of RuBPC - rpl2 gene for ribosomal protein L2 - rpoA gene for the subunit of RNA polymerase - rps11, rps12 and rps19 genes for ribosomal proteins S11, S12, and S19, respectively - rps19 open reading frame for a protein with N terminus similar to that of S19 - RuBPC ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) - trnH gene for histidine transfer RNA - URF39 and URF509 unidentified reading frames for polypeptides of 39 and 509 amino acids, respectively Gene names follow the convention of Hallick and Bottomley (1983)     

Organization of the psbE,psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome

Summary The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE — 8 by spacer —psbF — 110 by spacer — orf38 — 87 by spacer — orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T > A > G > C in the mRNA-like strand and group II-like boundary sequences at their 3 and 5 ends having the consensus 5-GTGTG .. INTRON .. TTAATTTNAT-3. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE psbF orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.Abbreviations and notations DEP diethyl pyrocarbonate; gene names follow the convention of Hallick and Bottomley (1983) - psaA, psaB genes for the P₇₀₀ apoproteins - psbE and psbF genes for the subunits of cytochrome b ₅₅₉ - orfN open reading frame of N codons     

Chloroplast DNA differences in the genus Oenothera subsection Munzia: a short direct repeat resembling the lambda chromosomal attachment site occurs as a deletion/insertion within an intron of an NADH-dehydrogenase gene

Summary A small restriction fragment length mutation has been mapped in the large inverted repeats of the chloroplast (cp) DNA of Munzia-Oenothera species (vom Stein and Hachtel 1986). This mutation could be localized within the intron of a reading frame presumably coding for subunit B of an NADH-dehydrogenase (ndhB). Sequence analysis revealed a 24 by duplication/deletion. The predicted secondary structure of the ndhB-intron is altered by this duplication/deletion. Part of the directly repeated segment shows remarkable similarity to the phage lambda attachment site. Evidence is presented for similar sequences in other plastome regions where deletions/insertions have been found. Furthermore, the locations of the genes for other components of the NADH-dehydrogenase (ndhA, ndhC, ndhD, ndhE, ndhF) were established by heterologous hybridization using gene probes from tobacco cpDNA.Abbreviations bp base pairs - kB 1,000 bp - cpDNA chloroplast DNA - Oe Oenothera - att-lambda attachment site of bacterio-lambda     

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

Summary We have determined the complete nucleotide sequence of the two genes encoding the P₇₀₀ chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5 GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3 and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains. Abbreviations and notations: Gene names follow the convention of Hallick and Bottomley (1983: psaA, psaB, genes for the P₇₀₀ apoprotein; psbE an psbF, genes foe the subunits of cytochrome b ₅₅₉; orfN, open reading frame of N condons     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

Non-random plastid segregation in somatic hybrids of Datura innoxia with several other Solanaceous species

Summary A non-random plastid segregation was found in somatic hybrids of Datura innoxia with seven different Solanaceous species. 14 out of 17 examined somatic hybrids showed the plastid features of Datura innoxia. Within the limits of sensitivity of the applied methods, one line could be shown to contain mixed plastids. Since sexual offspring of this line contains only one set of plastids, it is assumed that this is probably a periclinal chimaera due to the plastome, i.e., the plastid mixture is present on a plant rather than a cell level.